There is substantial interest in gene editing as a potential means to treat human genetic disorders such as thalassemia and sickle cell disease. Much effort has been focused on targeted nucleases such as CRISPR/Cas9 and zinc-finger nucleases (ZFNs), based on work showing that site-directed DNA damage strongly promotes homologous recombination (HR). However, clinical application of targeted nucleases is challenged by the risk of off-target cleavage events in the genome. As an alternative, in work recently published in Nature Communications, the Ly, Saltzman, and Glazer labs have shown that ?-substituted triplex- forming peptide nucleic acids (PNAs) and donor DNAs delivered intravenously (IV) via poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) into a mouse model of human ?-thalassemia produced almost complete amelioration of the disease, with clinically relevant ?-globin gene correction frequencies in hematopoietic stem cells (HSCs) of up to 7%. The mice showed alleviation of anemia, improvement in RBC morphologies, and reversal of splenomegaly and extramedullary hematopoiesis, with extremely low off-target effects in the genome, a key advantage of this technology. The other key advantage is that the components can be synthesized chemically and formulated into nanoparticles for simple IV administration. However, synthesis of ?PNAs is complicated and expensive, and they are not commercially available, limiting the ability of investigators to exploit this technology. In line with RFA-RM-18-024, ?Expanding the Human Genome Engineering Repertoire?, this multi-PI proposal by Ly, Saltzman, and Glazer seeks to advance PNA/NP-based gene editing by simplifying and scaling up PNA synthesis, by incorporating next generation PNA chemistry to boost binding affinity, increase selectivity, and enhance potency, and by strategically exploiting cellular DNA repair pathways. The Specific Aims are: (1) To scale up PNA production and augment DNA binding, in order to expedite the translation of PNAs for therapeutic gene editing and enable widespread adoption of the technology. We will devise an enantioselective strategy for scaling up the production of monomers, and we will synthesize and test ?PNAs with modified nucleobases to achieve improved DNA binding properties and to overcome the homopurine sequence restriction for triplex formation. (2) To develop strategies to manipulate DNA repair to enhance the efficiency of PNA-mediated gene editing, based on promising preliminary results with a novel DNA repair inhibitor. (3) To provide a robust platform of assays to evaluate the advancements from Aims 1-2 and to generalize this approach to multiple genes. We will continue to exploit facile mouse- and cell-based assays for correction of the human ?-globin gene at the IVS2-654 thalassemia mutation. We expect this work to provide the basis for designing even more potent PNAs applicable to gene editing for many human genetic disorders.